One of the most frequently used molecular biological techniques for detecting homologous nucleic acid sequences is DNA/DNA, RNA/RNA, or RNA/DNA hybridization. In this technique, nucleic acid (DNA or RNA) used as a probe is labeled, and the labeled nucleic acid is hybridized to a nucleic acid (DNA or RNA) to be detected. When the nucleic acid used as a probe has a homology to the nucleic acid to be detected, each single-stranded nucleic acid hybridizes to its complementary sequence so as to form a double-stranded sequence, and then the double-stranded sequence is detected by a label of the probe.
Conventionally, when nucleic acid is used as a probe, a technique of labeling the probe with radioisotope has been employed and the presence of hybridization between the probe and a target nucleic acid has been detected by autoradiography.
Although the technique using radioisotopes for labeling a gene probe is especially superior in its high sensitivity, there exist such problems that the handling of radioisotopes is complicated because safety of the laboratory must be ensured and special care must be taken in the disposal of radioactive wastes. Moreover, radioisotopes can be used only for a limited time because they have a half-life period.
For the abovementioned reasons, non-radioactive labeling techniques have been developed as more simple techniques. For example, techniques of labeling a gene probe with biotin molecules (European Patent No. 0 063 879) or with digoxigenin molecules (European Patent Application No. 0 324 474 A1) are known. After hybridization of a labeled nucleic probe to the nucleic acid sequence to be detected, biotin molecules or digoxigenin molecules are present in the resulting double-stranded nucleic acid. After hybridization, binding of (strept)avidin-marker enzyme complex or anti-digoxigenin antibody-marker enzyme complex to the resultant double-stranded nucleic acid sequence allows detection of nucleic acids to which the probes were hybridized. However, such detection methods using enzymes are insufficient in terms of sensitivity and specificity.
Other than the above techniques, various techniques of labeling a target substance with fluorescent dye have been studied. A desired fluorescent labeling reagent (1) possesses a high fluorescent quantum yield, (2) possesses a molecular absorption coefficient, (3) is water-soluble and does not self-quench by agglutinating in an aqueous solvent, (4) is not susceptible to hydrolysis, (5) does not photo-dissociate easily, (6) is not susceptible to background fluorescence, and (7) has a previously introduced reactive substituent which forms covalent binding with a target substance.
Fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate, which are well-known as fluorescent labeling reagents, possess high fluorescent quantum yields, but have drawbacks such that the molecular absorption coefficients are low and the excitation and luminous wavelength is 500 nm to 600 nm and therefore these reagents are susceptible to the influence of background fluorescence of a membrane used for blotting.
As dyes having a high molecular absorption coefficient, for example, polymethine dyes are known such as cyanine dye described in U.S. Pat. No. 5,486,616, Japanese Patent Application Laid-Open Nos. 2-191674, 5-287209, 5-287266, 8-47400, 9-127115, 7-145148 and 6-222059, and barbiturate oxonol described in Journal of Fluorescence, 5, 231, 1995. However, there exist some problems such that they are almost insoluble in water and if they are dissolved, hydrolysis occurs. Also, strong intermolecular interactions between dyes can cause formation of aggregates in an aqueous medium so that self-quenching of fluorescence is often observed.
Moreover, cyanine dyes described in Japanese Patent Application Laid Open No. 2-191674 and the like are superior dyes because they have water-solubility due to introduction of a sulfonic acid group into a relatively stable chromophore and the formation of aggregates is prevented. However, there exist some problems such that uptake efficiency of fluorescent nucleotides is poor by synthetic reactions of nucleic acids, for example, reverse transcription reaction.